A signal-on fluorosensor based on quench-release principle for sensitive detection of antibiotic rapamycin.

Jeong HJ, Itayama S, Ueda H - Biosensors (Basel) (2015)

Bottom Line:
We constructed rapamycin Q'-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin.The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system.As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q'-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.

ABSTRACTAn antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose "Q'-body", which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q'-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q'-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.

Mentions:
When we performed denaturation-induced de-quenching of ATTO520-labeled *RK Q’-body with 7 M GdnHCl and 100 mM DTT, the fluorescence intensity increased to ~4.9-fold (Figure A1). In other words, the dye is first quenched to ~20%, and possible maximum de-quenching of this Q’-body will be ~4.9-fold. However, the currently observed rapamycin-dependent de-quenching was modest 1.5-fold. Hence, to attain higher fluorescent response, the improvement of rapamycin-induced de-quenching efficiency will be the most effective strategy.

Mentions:
When we performed denaturation-induced de-quenching of ATTO520-labeled *RK Q’-body with 7 M GdnHCl and 100 mM DTT, the fluorescence intensity increased to ~4.9-fold (Figure A1). In other words, the dye is first quenched to ~20%, and possible maximum de-quenching of this Q’-body will be ~4.9-fold. However, the currently observed rapamycin-dependent de-quenching was modest 1.5-fold. Hence, to attain higher fluorescent response, the improvement of rapamycin-induced de-quenching efficiency will be the most effective strategy.

Bottom Line:
We constructed rapamycin Q'-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin.The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system.As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q'-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.

ABSTRACTAn antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose "Q'-body", which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q'-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q'-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.